Reversible inhibitors of S-adenosyl-L-homocysteine hydrolase and uses thereof

ABSTRACT

The present invention provides compositions and methods for reversibly inhibiting S-adenosyl-L-homocysteine (SAH) hydrolase. The compounds of the present invention can be used in combination with an anti-hemorrhagic viral infection agent, an immunosuppressant, a homocysteine lowering agent, or an anti-neoplasm agent. The compositions and methods of the present invention can be used for the prevention and treatment of hemorrhagic virus infection, autoimmune diseases, autograft rejection, neoplasm, hyperhomocysteineuria, cardiovascular disease, stroke, Alzheimer&#39;s disease, or diabetes.

BACKGROUND OF THE INVENTION

SAH hydrolase has been an attractive target for antiviral drug designbased on the observation that many viruses require 5′-capped, methylatedstructures on their mRNA for efficient translation of viral proteins.Yuan et al., Exp. Opin. Ther. Patents, 9: 1197-1206 (1999); Yuan et al.,in Adv. Antiviral Drug Des. vol 2, pp. 41-88, De Clercq (ed)., JAIPress, Inc. London, UK (1996). Inhibition of SAH hydrolase results ininhibition of S-adenosyl-L-methionine (SAM)-dependent methylationreactions, including viral mRNA methylation, thus inhibiting viralreplication (Scheme 1).

Numerous inhibitors of SAH hydrolase have been identified from naturallyoccurring compounds and synthetic compounds. Most potent inhibitors areirreversible inhibitors, which irreversibly inactivate SAH hydrolase ina time-dependent fashion. Studies have demonstrated that irreversibleinhibitors only produce narrow therapeutic windows due to their severecytotoxic effects (Wolfe and Borchardt, Journal of Medicinal Chemistry,34:1521-1530 (1991)). Since SAH hydrolase is a ubiquitous cellularenzyme with a very slow turnover rate (t_(1/2)=24 hours in mouse liver),irreversible inhibitors can cause prolonged inhibition of the enzymeactivity. For instance, it can take up to seven days for completerecovery of enzyme activity, which can lead to unwanted side effects.The severe cytotoxicity associated with irreversible inhibitors has beenthe major factor that has impaired the development of these inhibitorsinto clinically useful drugs. Because of the cytotoxicity associatedwith irreversible inhibitors, reversible inhibitors are preferred.

However, at present, there are no known reversible SAH hydrolaseinhibitors that are potent enough to produce substantial inhibitoryactivity against the enzyme when tested in vivo. For example, thereversible inhibitor (S)-9-(2,3-dihydroxypropyl)adenine ((S)-DHPA),which has a K_(i) value of 3.5 μM against SAH hydrolase, lacksinhibitory potency. (Votruba and Holy, Coll. Czech. Chem. Commun.,45:3039 (1980)). Though (s)-DHPA was reported to be a reversibleinhibitor of isolated AdoHcy hydrolase (Votruba and Holy, Coll. Czech.Chem. Commun., 45:3039 (1980)), it was also reported to be airreversible inhibitor of intracellular AdoHcy hydrolase (Schanche etal., Molecular Pharmacology, 26:553-558 (1984)). Thus, existingreversible inhibitors are not clinically useful therapeutic agents, andthere remains a need for SAH hydrolase inhibitors that exhibit potencywithout the undesired cytotoxic effects.

BRIEF SUMMARY OF THE INVENTION

The present invention provides novel reversible inhibitors of SAHhydrolase. The compounds of the present invention are useful as agentsdemonstrating biological activities related to their ability to inhibitSAH hydrolase. In one embodiment, the reversible inhibitors of SAHhydrolase have the formula (I), and pharmaceutically acceptable saltsthereof:

wherein Z is carbon or nitrogen, R1 and R2 are the same or different,and are hydrogen, hydroxy, alkyl, cycloalkyl, alkenyl, alkoxy, amino,aryl, heteroaryl, or halogen; R3 and R4 are the same or different andare hydrogen, alkyl, acetyl, alkenyl, aryl, or heteroaryl; X is oxygen,nitrogen, or sulfur; and Y is hydrogen, a C₁₋₁₀ alkyl group, alkenyl,vinyl, aryl, or heteroaryl. In a particular embodiment, the compound isnot (4-adenine-9-yl)-2-hydroxybutanoic acid.

Compounds of formula I can have an S configuration at the β carbon, an Rconfiguration at the β carbon, or comprise a racemic mixture. In oneembodiment, the compounds have a K_(i) value less than 100 nM for amammalian SAH hydrolase in a biological medium, e.g., serum. In otherembodiments, the compounds have a K_(i) value between about 1 nM andabout 100 nM for a mammalian SAH hydrolase in a biological medium. Thecompounds preferably have a K_(i) value less than 100 nM, or a K_(i)value between about 1 nM and about 100 nM for a human SAH hydrolase in abiological medium.

Compounds of formula I can have substituents wherein R1, R2, R3, and R4are hydrogen. In one aspect of the present invention, X is oxygen. Inanother aspect of the present invention, Y is hydrogen or a C₁₋₁₀ alkylgroup. In yet another aspect of the present invention, R1, R2, R3, andR4 are hydrogen, X is oxygen, and Y is hydrogen or a C₁₋₁₀ alkyl group.

The present invention also relates to a pharmaceutical compositioncomprising an effective amount of a compound of formula I orpharmaceutically acceptable salts thereof, and a pharmaceuticallyacceptable carrier or diluent. Pharmaceutical compositions may beadministered by oral, parenteral (e.g., intramuscular, intraperitoneal,intravenous, intracisternal injection or infusion, subcutaneousinjection, or implant), inhalation spray, nasal, vaginal, rectal,sublingual, or topical routes of administration. The pharmaceuticalcompositions may be formulated in suitable dosage unit formulationsappropriate for each route of administration.

It is not intended that the present invention be limited to particularformulations or particular modes of administration. In one embodiment,the composition is formulated for oral, parenteral, intranasal, topical,or injectable administration. Non-limited examples of injectableadministration are intracavernous injection, subcutaneous injection,intravenous injection, intramuscular injection and intradermalinjection. The pharmaceutical composition can be formulated for oraladministration in a dosage ranging from about 0.1 to about 20 mg/kg perday. The pharmaceutical composition can also be formulated forinjectable administration in a dosage ranging from about 0.1 to about 20mg/kg per day.

Pharmaceutical compositions of the present invention can be formulatedin a solid or liquid dosage form. For example, the pharmaceuticalcompositions may be formulated as a solid in the form of tablets,capsules, granules, powders, and similar compounds. The pharmaceuticalcompositions may also be formulated as a liquid in the form of syrups,injection mixtures, and the like.

The present invention also provides a kit comprising an effective amountof the composition of the present invention, and an instruction meansfor administering the composition.

Furthermore, the present invention provides methods for reversiblyinhibiting the activity of a S-adenyl-L-homocysteine (SAH) hydrolase. Inone embodiment, the present invention provides a method for reversiblyinhibiting activity of a S-adenosyl-L-homocysteine (SAH) hydrolase in amammal, comprising administering to a mammal to which such reversibleinhibition is needed or desirable, an effective amount of a compound ora pharmaceutically acceptable salt thereof, having the formula (I):

wherein Z is selected from the group consisting of carbon and nitrogen,R1 and R2 are the same or different, and are selected from the groupconsisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkenyl, alkoxy,amino, aryl, heteroaryl, and halogen; R3 and R4 are the same ordifferent and are selected from the group consisting of hydrogen, alkyl,acetyl, alkenyl, aryl, and heteroaryl; X is selected from the groupconsisting of oxygen, nitrogen, and sulfur; and Y is selected from thegroup consisting of hydrogen, a C₁₋₁₀ alkyl group, alkenyl, vinyl, aryl,and heteroaryl, thereby reversibly inhibiting the activity of SAHhydrolase in said mammal. In a particular embodiment, the administeredcompound or a pharmaceutically acceptable derivative thereof is not(4-adenine-9-yl)-2-hydroxybutanoic acid.

In preferred embodiments, the mammal is suspected of having a diseaseselected from the group consisting of hemorrhagic viral infection,autoimmune disease, autograft rejection; neoplasm,hyperhomocysteineuria, cardiovascular disease, stroke, Alzheimer'sdisease, diabetes, inflammatory Bowel disease, multiple sclerosis andautoimmune neuritis. However, it is not intended that the presentinvention be limited to the prevention and treatment of particulardiseases.

It is an object of the present invention to provide methods forpreventing and treating hemorrhagic viral infections. In one aspect, themethod comprises administering an effective amount of compounds havingformula I in the treatment of hemorrhagic viral infections in a mammal.In particular embodiments, the hemorrhagic viral infection is caused bya virus selected from the group consisting of a Bunyaviridaea, aFiloviridae, a Flaviviridae, and an Arenaviridae virus. In otherparticular embodiments; the Filoviridae virus is Ebola virus.

It is also an object of the present invention to provide methods forpreventing and treating autoimmune diseases. In one aspect, the methodcomprises administering an effective amount of compounds having formulaI in the treatment of an autoimmune disease in a mammal.

It is also an object of the present invention to provide methods forpreventing and treating allograft rejection. In one aspect, the methodcomprises administering an effective amount of compounds having formulaI in the treatment of allograft rejection in a mammal.

Furthermore, it is an object of the present invention to provide methodsfor preventing or treating hyperhomocysteineuria, or for lowering plasmahomocysteine in a mammal. In one aspect, the method comprisesadministering an effective amount of compounds having formula I forlowering plasma homocysteine in a mammal.

Further, it is an object of the present invention to provide methods forpreventing or treating neoplasm. In one aspect, the method comprisesadministering an effective amount of compounds having formula I for inthe treatment of neoplasm in a mammal. Non-limiting examples of neoplasmare neoplasm of the adrenal gland, anus, auditory nerve, bile ducts,bladder, bone, brain, breast, bruccal, central nervous system, cervix,colon, ear, endometrium, esophagus, eye, eyelids, fallopian tube,gastrointestinal tract, head and neck, heart, kidney, larynx, liver,lung, mandible, mandibular condyle, maxilla, mouth, nasopharynx, nose,oral cavity, ovary, pancreas, parotid gland, penis, pinna, pituitary,prostate gland, rectum, retina, salivary glands, skin, small intestine,spinal cord, stomach, testes, thyroid, tonsil, urethra, uterus, vagina,vestibulocochlear nerve, and the vulva.

The present invention also provides a combination, comprising aneffective amount of a compound having formula I, and an effective amountof an anti-hemorrhagic viral infection agent, an immunosuppressant, aplasma homocysteine lowering agent, and an anti-neoplasm agent. Thecombination can further comprise a pharmaceutically acceptable carrieror excipient. In a particular embodiment, the combination does notinclude (4-adenine-9-yl)-2-hydroxybutanoic acid.

In a particular embodiment, the anti-hemorrhagic viral infection agentinhibits interleukin-1 (IL-1), tumor necrosis factor (TNF), or acombination thereof. The anti-hemorrhagic viral infection agent can bean anti-viral vaccine, an anti-viral antibody, a viral-activated immunecell, or a viral-activated immune serum.

In another embodiment, the immunosuppressant is cyclosporine,tacrolimus, an adrenocortical steroid, azathioprine, mycophenolate,cyclophosphamide, methotrexate, chlorambucil, vincristine, vinblastine,dactinomycin, an antithymocyte globulin, muromonab-CD3 monoclonalantibody, Rh₀(D) immunoglobulin, methoxsalen, or thalidomide.

In other particular embodiments, the homocysteine lowering agent isvitamin B₆, vitamin B₁₂, or folate.

In yet other embodiments, the anti-neoplasm agent is an anti-angiogenicagent, an alkylating agent, an antimetabolite, a natural product, aplatinum coordination complex, an anthracenedione, a substituted urea, amethylhydrazine derivative, an adrenocortical suppressant, a hormone, anantagonist, an oncogene inhibitor, a tumor suppressor gene or protein,an anti-oncogene antibody, or an anti-oncogene antisenseoligonucleotide.

The present invention also provides a kit comprising an effective amountof the combination of the present invention, and an instruction meansfor administering the combination.

Furthermore, the present invention provides a method for reversiblyinhibiting activity of a SAH hydrolase in a mammal, comprisingadministering to a mammal to which such reversible inhibition is neededor desirable, an effective amount of a combination, wherein thecombination comprises: a) an effective amount of a compound or apharmaceutically acceptable salt thereof, having the formula (I):

wherein Z is selected from the group consisting of carbon and nitrogen,R1 and R2 are the same or different, and are selected from the groupconsisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkenyl, alkoxy,amino, aryl, heteroaryl, and halogen; R3 and R4 are the same ordifferent and are selected from the group consisting of hydrogen, alkyl,acetyl, alkenyl, aryl, and heteroaryl; X is selected from the groupconsisting of oxygen, nitrogen, and sulfur; and Y is selected from thegroup consisting of hydrogen, a C₁₋₁₀ alkyl group, alkenyl, vinyl, aryl,and heteroaryl; and b) an effective amount of a compound selected fromthe group consisting of an anti-hemorrhagic viral infection agent, animmunosuppressant, a homocysteine lowering agent, and an anti-neoplasmagent, thereby reversibly inhibiting said activity of SAH hydrolase insaid mammal. In a particular embodiment, the administered combinationdoes not include (4-adenine-9-yl)-2-hydroxybutanoic acid.

The combination can be used with any other pharmaceutical composition tomodulate SAH hydrolase activity in a mammal. The combination can also beused in the prevention and treatment of diseases such as hemorrhagicviral infection, autoimmune disease, autograft rejection, neoplasm, andhyperhomocysteineuria, cardiovascular disease, stroke, Alzheimer'sdisease, diabetes, inflammatory Bowel disease, multiple sclerosis orautoimmune neuritis, as described above. However, it is not intendedthat the combination be limited to the prevention and uses of particulardiseases.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 illustrates effects of DZ2002 on Quantitative hemolysis of SheepRed Blood Cells (QHS) assay. Data were expressed as means±SD. **: P<0.01compared with control.

FIG. 2 illustrates that DZ2002 suppresses T cell proliferation in mixedlymphocyte reaction. Data were expressed as means±SD. ***: P<0.001compared with control.

FIG. 3 illustrates that DZ2002 have no cytotoxicity in spleen cell.

FIG. 4 illustrates effects of DZ2002 on DTH ear swelling in Balb/c mice.

FIG. 5 illustrates effects of DZ2002 on TNF-α production from TG inducedperitoneal cells.

FIG. 6A illustrates effects of DZ2002 on the expression of MHC-II onTHP-1 cells; FIG. 6B illustrates effects of DZ2002 on the expression ofCD80 on THP-1 cells; and FIG. 6C illustrates effects of DZ2002 on theexpression of CD86 on THP-1 cells.

FIGS. 7A and 7B illustrate effects of DZ2002 on IL-12P40 and IL-12P70production from THP-1 cells.

DETAILED DESCRIPTION OF THE INVENTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections thatfollow.

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entirety. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more.”

As used herein, a “composition” refers to any mixture of two or moreproducts or compounds. It may be a solution, a suspension, liquid,powder, a paste, aqueous, non-aqueous, or any combination thereof.

As used herein, a “combination” refers to any association between two oramong more items.

As used herein, “homocysteine” (Hcy) refers to a compound with thefollowing molecular formula: HSCH₂CH₂CH(NH₂)COOH. Biologically, Hcy isproduced by demethylation of methionine and is an intermediate in thebiosynthesis of cysteine from methionine. The term “Hcy” encompassesfree Hcy (in the reduced form) and conjugated Hcy (in the oxidizedform). Hcy can conjugate with proteins, peptides, itself or other thiolsthrough a disulfide bond.

As used herein, “SAH hydrolase” refers to an enzyme which catalyzeshydrolysis of SAH to adenosine (Ado) and Hcy. The enzyme is anubiquitous eukaryotic enzyme, which is also found in some prokaryotes.SAH hydrolase also catalyzes the formation of SAH from Ado and Hcy. Theco-enzyme of SAH hydrolase is NAD⁺/NADH. SAH hydrolase may have severalcatalytic activities. In the hydrolytic direction, the first stepinvolves oxidation of the 3′-hydroxyl group of SAH (3′-oxidativeactivity) by enzyme-bound NAD⁺(E-NAD⁺), followed by β-elimination of1-Hcy to give 3′-keto-4′,5′-didehydro-5′-deoxy-Ado. Michael addition ofwater to the 5′-position to this tightly bound intermediate(5′-hydrolytic activity) affords 3′-keto-Ado, which is then reduced byenzyme-bound NADH (E-NADH) to Ado (3′-reduction activity). It isintended to encompass SAH hydrolase with conservative amino acidsubstitutions that do not substantially alter its activity.

As used herein, the terms “pharmaceutically acceptable salts” or“pharmaceutically acceptable derivatives” of the compounds of thepresent invention encompass any salts, esters or derivatives that may bereadily prepared by those of skill in this art. Pharmaceuticallyacceptable salts of the compounds of this invention include, forexample, those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Salts derived from appropriate bases include,but are not limited to, alkali metal (e.g., sodium), alkaline earthmetal (e.g., magnesium), ammonium and N(C₁₋₄ alkyl)₄ ⁺ salts. Examplesof suitable acids include, but are not limited to, hydrochloric,hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric,acetic, citric, methanesulfonic, formic, benzoic, malonic,naphthalene-2-sulfonic, and benzenesulfonic acids. Other acids, such asoxalic, while not in themselves pharmaceutically acceptable, may beemployed in the preparation of salts useful as intermediates inobtaining the compounds of the invention and their pharmaceuticallyacceptable acid salts.

As used herein, “biological activity” refers to the in vivo activitiesof a compound or physiological responses that result upon in vivoadministration of a compound, composition, or other mixture. Biologicalactivity, thus, encompasses therapeutic effects and pharmaceuticalactivity of such compounds, compositions and mixtures. Biologicalactivities may be observed in in vitro systems designed to test or usesuch activities.

As used herein, “plasma” refers to the fluid, noncellular portion of theblood, distinguished from the serum obtained after coagulation.

As used herein, “serum” refers to the fluid portion of the bloodobtained after removal of the fibrin clot and blood cells, distinguishedfrom the plasma in circulating blood.

As used herein. “fluid” refers to any composition that can flow. Fluidsthus encompass compositions that are in the form of semi-solids, pastes,solutions, aqueous mixtures, gels, lotions, creams, and other suchcompositions.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are in accord with their common usage, recognizedabbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature,unless otherwise indicated (see Biochemistry 11: 1726 (1972)).

As used herein, “disease or disorder” refers to a pathological conditionin an organism, which is characterizable by identifiable symptoms.

As used herein, the term “a therapeutic agent” refers to anyconventional drug or drug therapies which are known to those skilled inthe art, including, but not limited to vaccines.

As used herein, “vaccine” refers to any compositions intended for activeimmunological prophylaxis. A vaccine may be used therapeutically totreat a disease, to prevent development of a disease, or to decrease theseverity of a disease either proactively or after infection. Exemplaryvaccines include, but are not limited to, preparations of killedmicrobes of virulent strains, living microbes of attenuated (variant ormutant) strains, or microbial, fungal, plant, protozoa, or metazoaderivatives or products. The term also encompasses protein/peptide andnucleotide based vaccines.

As used herein, the term “therapeutically effective amount” refers tothat amount that is sufficient to ameliorate, or in some manner reducethe symptoms associated with the disease. Such amount may beadministered as a single dosage or according to a regimen. Repeatedadministration may be required to achieve the desired amelioration ofsymptoms.

As used herein, the terms “administration” or “administering” a compoundrefers to any suitable method of providing a compound of the inventionor a pro-drug of a compound of the invention to a subject.

As used herein, the term “treatment” refers to any manner in which thesymptoms of a condition, disorder or disease are ameliorated orotherwise beneficially altered. Treatment also encompasses anypharmaceutical use of the compositions herein. Amelioration of symptomsof a particular disorder refers to any lessening of symptoms. whetherpermanent or temporary, that can be attributed to or associated withadministration of the composition.

As used herein, the term “substitute” refers to the replacement of ahydrogen atom in a compound with a substituent group.

As used herein, the term “alkyl” encompasses straight or branched alkylgroups, including alkyl groups that are optionally substituted with oneor more substituents. For example, the alkyl group can be optionallysubstituted with hydroxy, halogen, aryl, alkoxy, acyl, or othersubstituents known in the art. One of more carbon atoms of the alkylgroup can also be optionally replaced by one or more heteroatoms.

As used herein, the term “K_(i)” refers to a numerical measure of theeffectiveness of a compound in inhibiting the activity of a targetenzyme such as ICE. Lower values to K_(i) reflect higher effectiveness.The K_(i) value is derived by fitting experimentally determined ratedata to standard enzyme kinetic equations (Segel, Enzyme Kinetics,Wiley-Interscience, 1975).

As used herein, “an anti-neoplastic treatment” refers to any treatmentdesigned to treat the neoplasm, tumor or cancer by lessening orameliorating its symptoms. Treatments that prevent the occurrence orlessen the severity of neoplasm, tumor or cancer are also contemplated.

As used herein, “neoplasm (neoplasia)” refers to abnormal new growth,and thus means the same as tumor, which may be benign or malignant.Unlike hyperplasia, neoplastic proliferation persists even in theabsence of the original stimulus.

As used herein, “an anti-neoplasm agent (used interchangeably withanti-neoplastic agent, anti-tumor or anti-cancer agent)” refers to anyagents used in the anti-neoplasm treatment. These include any agents,that when used alone or in combination with other compounds, canalleviate, reduce, ameliorate, prevent, place or maintain in a state ofremission clinical symptoms or diagnostic markers associated withneoplasm, tumor or cancer. The anti-neoplasm agent that can be used inthe combinations of the present invention include, but are not limitedto, anti-angiogenic agents, alkylating agents, antimetabolite, certainnatural products, platinum coordination complexes, anthracenediones,substituted ureas, methylhydrazine derivatives, adrenocorticalsuppressants, certain hormones and antagonists, anti-cancerpolysaccharides, and certain herb extracts such as Chinese herbextracts.

As used herein, “tumor suppressor gene” (also referred to asanti-oncogene or cancer susceptibility gene) refers to a gene thatencodes a product which normally negatively regulates the cell cycle,and which must be mutated or otherwise inactivated before a cell canproceed to rapid division. Exemplary tumor suppressor genes include, butare not limited to, p16, p21, p53, RB (retinoblastoma), WT-1 (Wilm'stumor), DCC (deleted in colonic carcinoma), NF-1 (neurofibrosarcoma) andAPC (adenomatous polypospis coli).

As used herein, “oncogene” refers to a mutated and/or overexpressedversion of a normal gene of animal cells (the proto-oncogene) that in adominant fashion can release the cell from normal restraints on growth.Thus, an oncogene alone, or in concert with other changes, converts acell into a tumor cell. Exemplary oncogenes include, but are not limitedto, abl, erbA, erbB, ets, fes (fps), fgr, fms, fos, hst, int1, int2,jun, hit, B-lym, mas, met, mil (raf), mos, myb, myc, N-myc, neu (ErbB2),rat (mil), Ha-ras, Ki-ras, N-ras, rel, ros, sis, src, ski, trk and yes.

As used herein, “antisense polynucleotides” refer to synthetic sequencesof nucleotide bases complementary to mRNA or the sense strand of doublestranded DNA. Admixture of sense and antisense polynucleotides underappropriate conditions leads to the binding of the two molecules, orhybridization. When these polynucleotides bind to (hybridize with) mRNA,inhibition of protein synthesis (translation) occurs. When thesepolynucleotides bind to double stranded DNA, inhibition of RNA synthesis(transcription) occurs. The resulting inhibition of translation and/ortranscription leads to an inhibition of the synthesis of the proteinencoded by the sense strand.

As used herein, “antibody” includes antibody fragments, such as Fabfragments, which are composed of a light chain and the variable regionof a heavy chain.

As used herein, “humanized antibodies” refers to antibodies that aremodified to include “human” sequences of amino acids so thatadministration to a human will not provoke an immune response. Methodsfor preparing such antibodies are known. For example, the hybridoma thatexpresses the monoclonal antibody is altered by recombinant DNAtechniques to express an antibody in which the amino acid composition ofthe non-variable regions is based on human antibodies. Computer programshave been designed to identify such regions.

As used herein, “anti-hemorrhagic virus agent” or “anti-viralhemorrhagic agent” refer to any agent used in the treatment ofhemorrhagic viral infections. These include any agents, alone or incombination with other compounds, that can alleviate, reduce,ameliorate, prevent, or maintain in a place of remission clinicalsymptoms or diagnostic markers associated with viral hemorrhagicdiseases, or disorders. Non-limiting examples of antiviral-hemorrhagicagents include interleukin-1 (IL-1) inhibitors, tumor necrosis factor(TNF) inhibitors, anti-viral vaccines, anti-viral antibodies,viral-activated immune cells, and viral-activated immune sera.

As used herein, “an anti-hemorrhagic virus treatment” refers to anytreatment designed to treat hemorrhagic viral infections by lessening orameliorating the symptoms. Treatments that prevent the infection orlessen its severity are also contemplated.

As used herein, “IL-1 inhibitor” encompasses any substances that preventor decrease production, post-translational modifications, maturation, orrelease of IL-1, or any substances that interfere with or decrease theefficacy of the interaction between IL-1 and IL-1 receptor. Preferably,the IL-1 inhibitor is an anti-IL-1 antibody, an anti-IL-1 receptorantibody, an IL-1 receptor antagonist, an IL-1 production inhibitor, anIL-1 receptor production inhibitor, or an IL-1 releasing inhibitor.

As used herein, “tumor necrosis factor” (“TNF”) refers to a group ofproinflammatory cytokines encoded within the major histocompatibilitycomplex. The TNF family members include TNF a and TNFR (also known ascachectin and lymphotoxin, respectively). Complementary cDNA clonesencoding TNA α and TNFR have been isolated. Thus, reference to “TNF”encompasses all proteins encoded by the TNF gene family, including TNF αand TNF, or an equivalent molecule obtained from any other source orthat has been prepared synthetically. It is intended to encompass TNFwith conservative amino acid substitutions that do not substantiallyalter its activity.

As used herein, “TNF inhibitor” encompasses any substances that preventor decrease production, post-translational modifications, maturation, orrelease of TNF, or any substances that interfere with or decrease theefficacy of the interaction between TNF and TNF receptor. Preferably,the TNF inhibitor is an anti-TNF antibody, an anti-TNF receptorantibody, a TNF receptor antagonist, a TNF production inhibitor, a TNFreceptor production inhibitor, or a TNF releasing inhibitor.

B. Reversible Inhibitors of S-Adenosyl-L-Homocysteine Hydrolase

One approach for minimizing mechanism-based cytotoxicity is to optimizethe pharmacokinetic profiles of SAH hydrolase inhibitors, such that theinhibitors exhibit reversible inhibiting activity. Pharmacokineticprofiles can be optimized by optimizing K_(Off) values. For example,K_(Off) values are optimized such that they are small enough to producedesired antiviral effects, but large enough to allow adequate recoveryof the enzyme activity before the next dose.

Eritadenine Derivatives as Reversible Inhibitors of SAH Hydrolase

The present invention relates to novel inhibitors ofS-adenosyl-L-homocysteine compositions that are reversible and potent.For example, the present invention provides compounds with a K_(i) valueof less than 100 nM. In one embodiment, the present invention provides4(adenine-9-yl)-2-hydroxybutanoic acid, its derivatives, andpharmaceutically acceptable salts thereof, and methods for reversiblyinhibiting SAH hydrolase using such compounds.

The reversible inhibitor, 4(adenine-9-yl)-2-hydroxybutanoic acid issynthesized from deoxyl modification of eritadenine at the beta carbon.Eritadenine is a naturally occurring compound and a potent irreversibleinhibitor of SAH hydrolase. Deoxyl modification of eritadenine at thebeta carbon results in a compound that is a reversible inhibitor, whileretaining inhibitory potency. Derivatives of4(adenine-9-yl)-2-hydroxybutanoic acid can be synthesized usingconventional synthetic methods known to one of ordinary skill in theart. (See e.g., Yuan et al., Adv. Antiviral Drug Des. 2: 41-88 (1996);Holy et al., Coll. Czechoslovak Chem. Commun. 50: 245-279 (1985)).

Examples of 4(adenine-9-yl)-2-hydroxybutanoic acids derivatives include,but are not limited to, base-modified derivatives, and side-chainsubstituted derivatives. Base modified derivatives are derivatives of4(adenine-9-yl)-2-hydroxybutanoic acids with modifications at the adenylring base. The adenyl ring can be modified with various modifying groupsat the amino group. The adenyl ring can also be modified with varioussubstituents at the C2 and C8 positions of the adenyl ring.

In one embodiment, the reversible inhibitors of SAH hydrolase have thefollowing formula (I), and pharmaceutically acceptable salts thereof:

wherein Z is carbon or nitrogen, R1 and R2 are the same or different,and are hydrogen, hydroxy, alkyl, cycloalkyl, alkenyl, alkoxy, amino,aryl, heteroaryl, or halogen; R3 and R4 are the same or different andare hydrogen, alkyl, acetyl, alkenyl, aryl, or heteroaryl; X is oxygen,nitrogen, or sulfur; and Y is hydrogen, a C₁₋₁₀ alkyl group, alkenyl,vinyl, aryl, or heteroaryl.

The different R groups can be optionally substituted with othersubstituents. These substituents may be halogen, hydroxy, alkoxy, nitro,cyano, carboxylic acid, alkyl, alkenyl, cycloalkyl, thiol, amino, acyl,carboxylate, aryl, carbamate, carboxamide, sulfonamide, a heterocyclicgroup, or any appropriate substituent known in the art. In a particularembodiment, each R group is hydrogen, or a lower straight chain alkylsuch as methyl. In another embodiment, one or more carbon atoms in thealkyl or alkoxy groups may be replaced by one or more heteroatoms.

The amino group may also be substituted once or twice to form asecondary or tertiary amine. Non-limiting examples of substituentsinclude alkyls or an optionally substituted alkyl group; alkene or anoptionally substituted alkenyl group; cycloalkyl or an optionallysubstituted cycloalkyl group; aryl, heterocyclic; aralkyl (e.g. phenylC₁₋₄ alkyl); heteroalkyl such as phenyl, pyridine, phenylmethyl,phenethyl, pyridinylmethyl, pyridinylethyl; and other substituents. Theheterocyclic group may be a 5 or 6 membered ring containing 1-4heteroatoms.

The amino group may be substituted with an optionally substituted C₂₋₄alkanoyl (e.g. acetyl, propionyl, butyryl, isobutyryl etc.); a C₁₋₄alkylsulfonyl (e.g. methanesulfonyl, ethanesulfonyl, etc.); a carbonylor sulfonyl substituted aromatic or heterocyclic ring (e.g.benzenesulfonyl, benzoyl, pyridinesulfonyl, pyridinecarbonyl etc.).

The CO—X—Y group can be an optionally substituted carboxylate group.Examples of the optionally substituted carboxylate group include, butare not limited to, an optionally substituted alkyl (e.g. C₁₋₁₀ alkyl);an optionally substituted cycloalkyl (e.g. C₃₋₇ cycloalkyl); anoptionally substituted alkenyl (e.g. C₂₋₁₀ alkenyl); an optionallysubstituted cycloalkenyl (e.g. C₃₋₇ cycloalkenyl); an optionallysubstituted aryl (e.g. phenyl, naphthyl, C₁₋₄ aryl such as benzyl); andother appropriate substituents. Groups such as methoxymethyl,methoxyethyl, and related groups are also encompassed.

Structure-Based Drug Design of Novel SAH Hydrolase Inhibitors

It is also an object of the present invention to provide structure-baseddrug design using the compounds of the present invention as an initialtemplate molecule. Recently, X-ray structures of SAH hydrolase havebecome available for both “open” and “closed” forms of the enzyme. Usingstructure-based design, one of ordinary skill in the art can designnovel compounds for screening SAH hydrolase inhibitors. The design orselection of candidate compounds can begin with the selection of variousmoieties which fill binding pockets of the SAH hydrolase. (See e.g.,U.S. Pat. No. 5,756,466; Klebe, J. Mol. Med. 78: 69-281 (2000); andMaignan et al., Curr. Top. Med. Chem. 1: 161-174 (2001)).

There are a number of ways to select moieties to fill individual bindingpockets. These include visual inspection of a physical model or computermodel of the active site and manual docking of models of selectedmoieties into various binding pockets. Modeling software that is wellknown and available in the art can be used. These include, but are notlimited to, QUANTA (Molecular Simulations, Inc., Burlington, Mass.,1992); SYBYL (Molecular Modeling Software, Tripos Associates, Inc., St.Louis, Mo., 1992); AMBER (Weiner et al., J. Am. Chem. Soc. 6: 765-784(1984)); CHARMM (Brooks et al., J. Comp. Chem. 4: 187-217 (1983)). Themodeling step can be followed by energy minimization with standardmolecular mechanics forcefields such as CHARMM and AMBER. In addition,there are a number of more specialized computer programs to assist inthe process of selecting the binding moieties of this invention. Theseinclude, but are not limited to:

1. GRID (Goodford. “A Computational Procedure for DeterminingEnergetically Favorable Binding Sites on Biologically ImportantMacromolecules,” J. Med. Chem. 28: 849-857 (1985)). GRID is availablefrom Oxford University, Oxford, UK.

2. MCSS (Miranker et al., “Functionality Maps of Binding Sites: AMultiple Copy Simultaneous Search Method,” in Proteins. Structure,Function and Genetics” 11: 29-34 (1991)). MCSS is available fromMolecular Simulations, Burlington, Mass.

3. AUTODOCK (Goodsell et al., “Automated Docking of Substrates toProteins by Simulated Annealing,” in PROTEINS: Structure, Function andGenetics 8: 195-202 (1990)). AUTODOCK is available from the ScrippsResearch Institute, La Jolla, Calif.

4. DOCK (Kuntz et al., “A Geometric Approach to Macromolecule-LigandInteractions,” J. Mol. Biol. 161: 269-288 (1982)). DOCK is availablefrom the University of California, San Francisco, Calif.

Once suitable binding moieties have been selected, they can be assembledinto a single inhibitor. This assembly may be accomplished by connectingthe various moieties to a central scaffold. The assembly process may,for example, be done by visual inspection followed by manual modelbuilding, again using software such as QUANTA or SYBYL. A number ofother programs may also be used to help select ways to connect thevarious moieties. These include, but are not limited to:

1. CAVEAT (Bartlett et al., “CAVEAT: A Program to Facilitate theStructure-Derived Design of Biologically Active Molecules,” in MolecularRecognition in Chemical and Biological Problems, Special Pub., RoyalChem. Soc. 78: 182-196 (1989)). CAVEAT is available from the Universityof California, Berkeley, Calif.

2. 3D Database systems such as MACCS-3D (MDL Information Systems, SanLeandro, Calif.). This area has been recently reviewed by Martin(Martin, “3D Database Searching in Drug Design,” J. Med. Chem. 35:2145-2154 (1992)).

3. HOOK (available from Molecular Simulations, Burlington, Mass.)

In addition to the above computer assisted modeling of inhibitorcompounds, the inhibitors of this invention may be constructed de novousing either an empty active site or optionally including some portionsof a known inhibitor. Such methods are well known in the art. Theyinclude, for example:

1. LUDI (Bohm, “The Computer Program LUDI: A New Method for the De NovoDesign of Enzyme Inhibitors,” J. Comp. Aid. Molec. Design 6: 61-78(1992)). LUDI is available from Biosym Technologies, San Diego, Calif.

2. LEGEND (Nishibata et al., Tetrahedron, 47: 8985 (1991)). LEGEND isavailable from Molecular Simulations, Burlington, Mass.

3. LeapFrog (available from Tripos associates, St. Louis, Mo.).

A number of techniques commonly used for modeling drugs may be employed(see e.g., Cohen et al., J. Med. Chem. 33: 883-894 (1990)). Likewise anumber of examples in the chemical literature of techniques can beapplied to specific drug design projects. (For a review, see, Navia etal., Curr. Opin. Struc. Biol. 2: 202-210 (1991)). Using the novelcombination of steps of the present invention, the skilled artisan canadvantageously avoid time consuming and expensive experimentation todetermine enzymatic inhibition activity of particular compounds. Themethod is also useful in facilitating rational design of SAH hydrolaseinhibitors, and therapeutic and prophylactic agents against SAHhydrolase-mediated diseases. Accordingly, the present invention relatesto such inhibitors, and methods for identifying or selecting suchinhibitors.

A variety of conventional techniques may be used to carry out each ofthe above evaluations, as well as evaluations necessary in screening acandidate compound for SAH hydrolase inhibiting activity. Generally,these techniques involve determining the location and binding proximityof a given moiety, the occupied space of a bound inhibitor; thedeformation energy of binding of a given compound and electrostaticinteraction energies. Examples of conventional techniques useful in theabove evaluations include, but are not limited to, quantum mechanics,molecular mechanics, molecular dynamics, Monte Carlo sampling,systematic searches and distance geometry methods (Marshall, Ann. RefPharmacol. Toxicol. 27: 193 (1987)). Specific computer software has beendeveloped for use in carrying out these methods. Examples of programsdesigned for such uses include: Gaussian 92 (Gaussian, Inc., Pittsburgh,Pa.); AMBER; QUANTA/CHARMM; and Insight II/Discover (BiosysmTechnologies Inc., San Diego, Calif.). These programs may beimplemented, for instance, using a Silicon Graphics Indigo2 workstationor IBM RISC/6000 workstation model 550. Other hardware systems andsoftware packages will be known and be of evident applicability to thoseskilled in the art.

Different classes of active SAH hydrolase inhibitors, according to thisinvention, may interact in similar ways with the various binding pocketsof the SAH hydrolase active site. The spatial arrangement of theseimportant groups is often referred to as a pharmacophore. The concept ofthe pharmacophore has been well described in the literature (See Mayeret al., J. Comp. Aided Molec. Design 1: 3-16 (1987); Hopfinger et al. inConcepts and Applications of Molecular Similarity, Johnson and Maggiora(eds.), Wiley (1990))

Different classes of SAH hydrolase inhibitors of this invention may alsouse different scaffolds or core structures that allow the necessarymoieties to be placed in the active site such that the specificinteractions necessary for binding may be obtained. These compounds arebest defined in terms of their ability to match the pharmacophore, i.e.,their structural identity relative to the shape and properties of theactive site of SAH hydrolase. Various scaffolds have been described in,for example, Klebe, G., J. Mol. Med. 78: 269-281 (2000); Maignan et al.,Curr. Top. Med. Chem. 1: 161-174 (2001); and U.S. Pat. No. 5,756,466 toBemis et al.).

S-Adenosyl-L-Homocysteine Hydrolase to be Inhibited

The compounds of the present invention can be used to reversibly inhibitany SAH hydrolase. It is not intended that the present invention belimited to reversibly inhibiting any specific SAH hydrolase.

In one embodiment, the compounds of the present invention can be used toreversibly inhibit SAH hydrolase encoded by nucleic acids containingnucleotide sequences with the following GenBank accession Nos.: AF129871 (Gossypium hirsutum); AQ003753 (Cryptosporidium parvum); AF105295(Alexandrium fundyense); AA955402 (Rattus norvegicus); AA900229 (Rattusnorvegicus); AA874914 (Rattus norvegicus); AA695679 (Drosophilamelanogaster ovary); AA803942 (Drosophila melanogaster ovary; AI187655(Manduca sexta male antennae); U40872 (Trichomonas vaginalis); AJ007835(Xenopus Laevis); AF080546 (Anopheles gambiae); A1069796 (T. cruziepimastigote); Z97059 (Arabidopsis thaliana); AF059581 (Arabidopsisthaliana); U82761 (Homo sapiens); AA754430 (Oryza sativa); D49804(Nicotiana tabacum); D45204 (Nicotiana tabacum); X95636 (D.melanogaster); Ti 8277 (endosperm Zea mays); R75259 (Mouse brain);Z26881 (C. roseus); X12523 (D. discoideum); X64391 (Streptomycesfradiae); W21772 (Maize Leaf); AH003443 (Rattus norvegicus); U14963(Rattus norvegicus); U14962 (Rattus norvegicus); U14961 (Rattusnorvegicus); U14960 (Rattus norvegicus); U14959 (Rattus norvegicus);U14937 (Rattus norvegicus); U14988 (Rattus norvegicus); U14987 (Rattusnorvegicus); U14986 (Rattus norvegicus); U14985 (Rattus norvegicus);U14984 (Rattus norvegicus); U14983 (Rattus norvegicus); U14982 (Rattusnorvegicus); U14981 (Rattus norvegicus); U14980 (Rattus norvegicus);U14979 (Rattus norvegicus); U14978 (Rattus norvegicus); U14977 (Rattusnorvegicus); U14976 (Rattus norvegicus); U14975 (Rattus norvegicus);L32836 (Mus musculus); L35559 (Xenopus laevis); Z19779 (Human foetalAdrenals tissue); L23836 (Rhodobacter capsulatus); M15185 (Rat); L11872(Triticum aestivum); M19937 (Slime mold (D. discoideum); M80630(Rhodobacter capsulatus).

In another embodiment, the compounds of the present invention can beused to reversibly inhibit SAH hydrolase encoded by nucleic acidscontaining nucleotide sequences with the GenBank accession Nos.M61831-61832 (see also Coulter-Karis and Hershfield, Ann. Hum. Genet.,53(2):169-175 (1989)). The compounds of the present invention can alsobe used to reversibly inhibit SAH hydrolase encoded by nucleic acidscontaining the nucleotide or amino acid sequences set forth in U.S. Pat.No. 5,854,023.

C. Use as Therapeutic Agents

Reversible inhibition of SAH hydrolase using4(adenine-9-yl)-2-hydroxybutanoic acid, its derivatives, andpharmaceutically acceptable salts results in significantly reducedcytotoxicity while retaining its therapeutic effects. With its potencyand reversibility, the compounds of the present invention can be used astherapeutic agents without the severe toxicity associated with otherirreversible inhibitors. The compounds of the present invention areuseful as agents demonstrating biological activities related to theirability to inhibit SAH hydrolase. The inhibitory effect on SAH hydrolasecan be evaluated using the ratio of the initial rates of SAH hydrolysisin the presence or absence of the inhibitor, or using any methods knownto one of ordinary skill in the art. The present invention providescompositions and methods for the prevention and treatment of diseasessuch as hemorrhagic viral infection, autoimmune disease, autograftrejection, neoplasm, hyperhomocysteineuria, cardiovascular disease,stroke, Alzheimer's disease, and diabetes. However, it is not intendedthat the present invention be limited to the prevention and treatment ofparticular diseases.

1. Hemorrhagic Fever Viruses

The present invention provides compositions and methods for thetreatment of viral hemorrhagic fever. The reversible inhibitors of thepresent invention can serve as a broad-spectrum antiviral agent againstall types of viruses causing hemorrhagic fever, including, but notlimited to, togavirus, arenavirus, nairovirus, and hantavirus.Broad-spectrum antiviral drugs offer many advantages overnarrow-spectrum agents. Because of the difficulty associated withclinical diagnoses of viral pathogens, diagnostic results often arrivetoo late for the choice of a specific antiviral drug. Immediate actionis often necessary to prevent the condition of the patient fromworsening, particularly in acute infections where viral chemotherapymust start as soon as the patient presents clinical symptoms.

Inhibitors of S-adenosyl-L-homocysteine (SAH) hydrolase have beenreported to be effective in the treatment of Ebola viral infections. Thecompounds of the present invention can also be used against otherhemorrhagic diseases, such as those described in WO 00/64479. Althoughthe mechanism of inhibition is not necessary in practicing the methodsof the present invention, the mechanism of action by which the compoundsof the present invention inhibit viral replication may be based oninhibition of viral methylation.

2. Autoimmune Diseases and Diseases Associated with Immunosuppression

The present invention contemplates compositions and methods forpreventing and treating autoimmune diseases. If a person has anautoimmune disease, the immune system mistakenly attacks the cells,tissues, and organs of a person's own body. As a group, autoimmunediseases afflict millions of Americans. Most autoimmune diseases strikewomen more often than men. Examples of autoimmune diseases can be foundfrom the National Institute of Health, “Understanding AutoimmuneDisease”(http://www.niaid.nih.gov/publications/autoimmune/autoimmune.htm.).

Compounds that modulate SAH hydrolase activity may also be used for thetreatment of diseases that are associated with immunosuppression.Immunosuppression can be due to chemotherapy, radiation therapy,enhanced wound healing, enhanced burn treatment, or other drug therapysuch as corticosteroid therapy, or a combination of drugs used in thetreatment of autoimmune diseases and graft/transplantation rejection.Immunosuppression can also be due to congenital deficiency in receptorfunction, infectious diseases, parasitic diseases, or other causes.

3. Neoplasm and Cancer

The present invention also contemplates compositions and methods forpreventing and treating neoplasms, including, but not limited toneoplasm associated with the adrenal gland, anus, auditory nerve, bileducts, bladder, bone, brain, breast, bruccal, central nervous system,cervix, colon, ear, endometrium, esophagus, eye, eyelids, fallopiantube, gastrointestinal tract, head, neck, heart, kidney, larynx, liver,lung, mandible, mandibular condyle, maxilla, mouth, nasopharynx, nose,oral cavity, ovary, pancreas, parotid gland, penis, pinna, pituitary,prostate gland, rectum, retina, salivary glands, skin, small intestine,spinal cord, stomach, testes, thyroid, tonsil, urethra, uterus, vagina,vestibulocochlear nerve, vulva, and neoplasm associated with otherorgans. In particular embodiments, the pharmaceutical compositions ofthe present invention are useful for the treatment of non-small celllung cancer, lung cancer, breast cancer, and prostate cancer. Thepresent invention further contemplates compositions and methods forpreventing and treating cancers, including, but not limited to thoseassociated with solid tumors, lymphoma, metastatic tumors, glioblastomatumors, and other carcinomas tumors.

4. Diseases Associated with Increased Homocysteine Levels

Furthermore, it is contemplated that the compounds of the presentinvention can be used as a plasma homocysteine lowering agent for theprevention and treatment of diseases associated with increased levels ofhomocysteine. Diseases which have been found to be linked with increasedhomocysteine levels (i.e., hyperhomocysteinemia) include, but are notlimited to cardiovascular diseases, stroke, Alzheimer's disease anddiabetes. For example, various studies have shown a relation betweenhyperhomocysteinemia and coronary heart disease (CHD), peripheralvascular disease, stroke, and venous thrombosis.

The increased risk of stroke from high homocysteine levels also increasethe chance of developing Alzheimer's disease. Recent studies have alsoshown that people with dementia of the Alzheimer's type have elevatedlevels of homocysteine in their blood. (Selhub et al., “Plasmahomocysteine as a risk factor for dementia and Alzheimer's disease,” N.Eng. J. Med. 46: 476-483 (2002)). Elevated homocysteine has also beenlinked to complications in diabetes, lupus, and other chronic diseases.

D. Pharmaceutical Compositions

Pharmaceutical compositions of the present invention comprise any of thecompounds of the present invention and pharmaceutically acceptable saltsthereof, alone or in combination with any pharmaceutically acceptablecarriers, adjuvant or vehicle. Acceptable compositions and methods fortheir administration that can be employed for use in this inventioninclude, but are not limited to those described in U.S. Pat. Nos.5,736,154; 6,197,801; 5,741,511; 5,886,039; 5,941,868; 6,258,374 and5,686,102. Examples of pharmaceutically acceptable carriers, adjuvantsand vehicles that can be used in the pharmaceutical compositions of thisinvention include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

The formulation, dosage and route of administration can be determinedaccording to methods known in the art (see e.g., Remington: The Scienceand Practice of Pharmacy, Alfonso R. Gennaro (Editor) Mack PublishingCompany, April 1997; Therapeutic Peptides and Proteins: Formulation,Processing, and Delivery Systems, Banga, 1999; and PharmaceuticalFormulation Development of Peptides and Proteins, Hovgaard and Frkjr(Ed.), Taylor & Francis, Inc., 2000; Biopharmaceutical Drug Design andDevelopment, Wu-Pong and Rojanasakul (Ed.), Humana Press. 1999). In thetreatment or prevention of conditions which require SAH hydrolasemodulation, an appropriate dosage level will generally be about 0.01 to500 mg per kg body weight per day. Preferably, the dosage level will beabout 0.1 to about 250 mg/kg per day. In more preferred embodiments, thedosage level will range from about 0.1 to about 20 mg/kg per day. Theappropriate dosage can be administered in single or multiple dose. Itwill be understood that the specific dose level and frequency of dosagefor any particular subject may be varied and will depend upon a varietyof factors, including the activity of the specific compound used, themetabolic stability and length of action of that compound, the age, bodyweight, general health, sex, diet, mode and time of administration, rateof excretion, drug combination, the severity of the particularcondition, and the patient undergoing therapy.

The pharmaceutical compositions of this invention can be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally, via an implanted reservoir, or any suitable form ofadministration. The term parenteral as used herein includessubcutaneous, intracutaneous, intravenous, intramuscular,intra-articular, intrasynovial, intrasternal, intrathecal, intralesionaland intracranial injection or infusion techniques. The most suitableroute in any given case will depend on the nature and severity of thecondition being treated and on the nature of SAH hydrolase inhibitorbeing used.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(e.g., Tween 80), and suspending agents. The sterile injectablepreparation may also be a sterile injectable solution or suspension in anon-toxic parenterally-acceptable diluent or solvent. For example, thepharmaceutical composition may be a solution in 1,3-butanediol. Otherexamples of acceptable vehicles and solvents that may be employed in thecompositions of the present invention include, but are not limited to,mannitol, water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or diglycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, and aqueous suspensions and solutions. Inthe case of tablets for oral use, commonly used carriers include, butare not limited to, lactose and corn starch. Lubricating agents, such asmagnesium stearate, can also be added. For oral administration in acapsule form, useful diluents include lactose and dried corn starch.When aqueous suspensions are administered orally, the active ingredientis combined with emulsifying and suspending agents. If desired, certainsweetening, flavoring, and coloring agents may be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient. In particularembodiments, the excipient is solid at room temperature but liquid atthe rectal temperature. Thus, the excipient will melt in the rectum torelease the active components. Such materials include, but are notlimited to, cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation. Forexample, such composition may be prepared as solutions in saline,employing benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons, and/or othersolubilizing or dispersing agents known in the art.

The pharmaceutical compositions of this invention may also beadministered topically. For topical application to the skin, thepharmaceutical composition may be formulated with a suitable ointmentcontaining the active components suspended or dissolved in a carrier.Carriers for topical administration of the compounds of this inventioninclude, but are not limited to, mineral oil, liquid petroleum, whitepetroleum, propylene glycol, polyoxyethylene polyoxypropylene compound,emulsifying wax and water. Alternatively, the pharmaceutical compositioncan be formulated with a suitable lotion or cream containing the activecompound suspended or dissolved in a carrier. Suitable carriers include,but are not limited to, mineral oil, sorbitan monostearate, polysorbate60, cetyl esters wax, cetaryl alcohol, 2-octyldodecanol, benzyl alcoholand water. The pharmaceutical compositions of this invention may also betopically applied to the lower intestinal tract by rectal suppositoryformulation or in a suitable enema formulation. Topically-transdermalpatches are also included in this invention.

The invention also provides kits for carrying out the therapeuticregimens of the invention. Such kits comprise therapeutically effectiveamounts of an SAH hydrolase inhibitor, alone or in combination withother agents, in pharmaceutically acceptable form. Preferredpharmaceutical forms include inhibitors in combination with sterilesaline, dextrose solution, buffered solution, or other pharmaceuticallyacceptable sterile fluid. Alternatively, the composition may belyophilized or desiccated. In this instance, the kit may furthercomprise a pharmaceutically acceptable solution, preferably sterile, toform a solution for injection purposes. In another embodiment, the kitmay further comprise a needle or syringe, preferably packaged in sterileform, for injecting the composition. In other embodiments, the kitfurther comprises an instruction means for administering the compositionto a subject. The instruction means can be a written insert, anaudiotape, an audiovisual tape, or any other means of instructing theadministration of the composition to a subject.

E. Combinations for Reversibly Inhibiting SAH Hydrolase Activity

The present invention also provides combinations and kits for reversiblyinhibiting SAH hydrolase activity. In one embodiment, the presentinvention provides a combination, comprising an effective amount of acompound having formula I; and an effective amount of ananti-hemorrhagic viral infection agent, an immunosuppressant, a plasmahomocysteine lowering agent, or an anti-neoplasm agent. The combinationcan further comprise a pharmaceutically acceptable carrier or excipient.In yet another aspect, the present invention provides a kit, comprisingan effective amount of the combination as described, and an instructionmeans for administering the combination to a subject.

Any agent that can alleviate or ameliorate clinical symptoms ordiagnostic markers associated with viral hemorrhagic diseases can beused in the combination of the present invention. Anti-viral therapeuticagents include, but are not limited to, anti-viral vaccines, anti-viralantibiotics, viral-activated immune cells and viral-activated immunesera. WO 00/64479 describes examples of anti-viral therapeutic agentsthat can be used in the combination of the present invention. Preferredembodiments are antiviral therapeutic agents that exhibit biologicalactivity against viral hemorrhagic diseases caused by infection of aBunyaviridaea, a Filoviridae, a Flaviviridae, or an Arenaviridae virus.

Any agent that suppresses the ability of the body's immune system tofight disease can be used in the combination of the present invention.Non-limiting examples of immunosuppressants are cyclosporine,prednisilone, azathioprine, tacrolimus, an adrenocortical steroid,mycophenolate, cyclophosphamide, methotrexate, chlorambucil,vincristine, vinblastine, dactinomycin, an antithymocyte globulin,muromonab-CD3 monoclonal antibody, Rh₀(D) immune globulin, methoxsalen,and thalidomide (See, Goodman & Gilman's The Pharmacological Basis ofTherapeutics, (9th Ed.) McGraw-Hill 1996, pages 1294-1304). Theimmunosuppressant can be taken as a combination of drugs. For example,most people start on a combination of drugs (e.g., cyclosporin,azathioprine, and prednisilone combination) after their transplant. Overa period of time, the doses of each drug and the number of drugs takenmay be reduced as the risks of rejection decline.

Any agent that lowers homocysteine levels can be used in the combinationof the present invention. Folic acid is known to be an effectivehomocysteine-lowering agent. Other homocysteine-lowering agents include,but are not limited to, betaine, trimethylglycin, cyanobalamin, andother B-group vitamins. The combination can also include anymulti-vitamin and mineral supplement for use in lowering homocysteine.Examples of multivitamin and mineral supplements that can be used in thecombinations of the present invention, include, but are not limited to,those described in U.S. Pat. Nos. 6,361,800; 6,353,003; 6,323,188;6,274,170; 6,210,686; 6,203,818; and 5,668,173.

Any anti-neoplasm agent can be used in the combination of the presentinvention. Examples of anti-neoplasm agents that can be used in thecompositions and methods of the present invention are described in U.S.Patent Application No. 2002/044919. In one embodiment, the anti-neoplasmagent used is an anti-angiogenic agent. The anti-angiogenic agent can bean inhibitor of basement membrane degradation, an inhibitor of cellmigration, an inhibitor of endothelial cell proliferation, and aninhibitor of three-dimensional organization and establishment ofpotency. Examples of such anti-angiogenic agent are illustrated inAuerbach and Auerbach, Pharmacol. Ther., 63: 265-311 (1994); O'Reilly,Investigational New Drugs, 15: 5-13 (1997); J. Nat'l Cancer Instit., 88:786-788 (1996); and U.S. Pat. Nos. 5,593,990; 5,629,327 and 5,712,291.In another embodiment, the anti-neoplasm agent used is an alkylatingagent, an antimetabolite, a natural product, a platinum coordinationcomplex, an anthracenedione, a substituted urea, a methylhydrazinederivative, an adrenocortical suppressant, a hormone, and an antagonist.

Other anti-neoplasm agents include, but are not limited to, cytidine,arabinosyladenine (araC), daunomycin, doxorubicin, methotrexate (MTX),fluorinated pyrimidines such as 5-fluorouracil (5-FU), hydroxyurea,6-mercaptopurine, plant alkaloids such as vincristine (VCR), VP-16 andvinblastine (VLB), alkylating agent, cisplatin, nitrogen Mustard,trisamine, procarbazine, bleomycin, mitomycin C, actinomycin D, or anenzyme such as L-Asparaginase. The anti-neoplasm agent can also be anoncogene inhibitor such as an anti-oncogene antibody or an anti-oncogeneantisense oligonucleotide. In another embodiment, the anti-neoplasticagent is a cellular matrix inhibitor such as an anti-cellular-matrixantibody or an anti-cellular-matrix antisense oligonucleotide. Forexample, antibodies and antisense oligonucleotides against caveolin-1,decorin, cadherins, catenins, integrins, and other cellular matrix orcellular matrix genes can be used.

In a specific embodiment, the combination further comprises a tumorsuppressor gene for combined intratumoral therapy and gene therapy. Thegene can be used in the form of naked DNA, complexed DNA, cDNA, plasmidDNA, RNA or other mixtures thereof as components of the gene deliverysystem. In another embodiment, the tumor suppressor gene is included ina viral vector. Any viral vectors that are suitable for gene therapy canused in the combination. For example, an adenovirus vector (U.S. Pat.No. 5,869,305), a simian virus vector (U.S. Pat. No. 5,962,274), aconditionally replicating human immunodeficiency viral vector (U.S. Pat.No. 5,888,767), retrovirus. SV40, Herpes simplex viral amplicon vectorsand vaccinia virus vectors can be used. In addition, the genes can bedelivered in a non-viral vector system such as a liposome wherein thelipid protects the DNA or other biomaterials from oxidation during thecoagulation.

F. Examples Example 1 Synthesis of Reversible Inhibitors of SAHHydrolase

¹H (Me₄Si) NMR spectra were determined with solution in CDCl₃ at 400MHz, ¹³C (Me₄Si) at 100.6 MHz unless otherwise noted. Mass spectra (MS)were obtained by atmospheric pressure chemical ionization (APCI)technique. Reagent grade chemicals were used. Solvents were dried byreflux over and distillation from CaH₂ under an argon atmosphere, exceptTHF, which was distilled from benzophenone and potassium. TLC wasperformed on Merck kieselgel 60-F₂₅₄ with MeOH/CHCl₃ (1:9) andEtOAc/MeOH (95:5) as developing systems, and products were detected with254 nm light. Merck kieselgel 60 (230-400 mesh) was used for columnchromatography.

Elemental analyses were determined by Galbraith Laboratories, Knoxville,Tenn. Spectral data for isolated compounds were consistent with reporteddata. (Holy, Coll. Czech. Chem. Commun. 43, 3444-3464 (1978); Holy etal., Coll. Czech. Chem. Commun. 50: 262-279 (1985); Japanese Patent69-50781; Chem. Abstr. 1972: 514811). The syntheses are schematicallyshown in Scheme 2.

9-(3,4-O-Isopropylidene-3,4-dihydroxybutyl)adenine (1)

¹H NMR δ 1.36 (s, 3, CH₃), 1.45 (s, 3, CH₃), 2.00-2.05 (m, 1, H2′),2.22-2.24 (m, 1, H2″), 3.57-3.59 (m, 1, H3′), 4.03-4.06 (m, 2, H4′,4″),4.34-4.43 (m, 2, H1′, 1″), 5.76 (br s, 2, NH₂), 7.86 (s, 1, H8), 8.38(s, 1, H2); MS (APCI) m/z 264 (100, MH⁺).

9-(3,4-Dihydroxybutyl)adenine (2)

A solution of 1 (110 mg, 0.18 mmol) in CF₃COOH/H₂O (9:1) (5 ml) wasstirred for 20 min at ˜0° C. Volatiles were evaporated, coevaporatedwith toluene (3×) and EtOH (2×) to give 2 (73 mg, 78%) aftercrystallization from EtOH with spectra data as reported.

9-(4-O-t-Butyldimethylsilyl-3,4-dihydroxybutyl)adenine (3)

TBDMS-Cl (186 mg, 1.23 mmol) and imidazole (168 mg, 2.46 mmol) wereadded to a stirred solution of 2 (250 mg, 1.12 mmol) in dry DMF (8 mL).The mixture was stirred at ambient temperature for 5 h, then reactionmixture was partitioned between EtOAc/NH₄Cl/H₂O. The water layer wasextracted with next portion of EtOAc. The combined organic phase waswashed (brine), dried (Na₂SO₄), evaporated and the residue was columnchromatographed (CHCl₃/MeOH; 97: 3) to give 3 (234 mg, 62%): ¹H NMR δ0.04 (s, 6,2×CH₃), 0.88 (s, 9, t-Bu), 1.81-1.89 (m, 1, H2′), 2.03-2.11(m, 1, H2″), 3.50-3.52 (m, 2, H4′,4″), 3.58-3.59 (m, 1, H3′), 4.32-4.45(m, 2, H1′, 1″), 6.10 (br s, 2, NH₂), 7.87 (s, 1, H8), 8.36 (s, 1, H2);¹³C NMR δ −5.0 & −4.9 (2×CH₃), 18.7 (t-Bu), 26.3 (t-Bu), 33.7 (C2′),40.9 (C 1′), 67.4 (C4′), 68.4 (C3′), 119.9 (C5), 141.4 (C8), 150.5 (C4),153.1 (C2), 155.8 (C6); MS (APCI) m/z 338 (100, MH⁺). Anal. Calcd forC₁₅H₂₇N₅O₂Si (337.50): C, 53.38; H, 8.06; N, 20.75.

9-[4-O-t-Butyldimethylsilyl-3-O-(1-ethoxyethyl)-3,4-dihydroxybutyl]adenine(4)

Ethyl vinyl ether (214 mg, 0.28 mL, 2.96 mmol) and pyridiniump-toluenesulfonate (15 mg, mmol) were added to a solution of 3 (250 mg,0.74 mmol) in dry CH₂Cl₂ (30 mL), and the mixture was stirred at ambienttemperature under N₂ until no starting material was detected by TLC(usually 5-6 days). Then reaction mixture was washed with water, dried(Na₂SO₄), and was evaporated. Column chromatography (EtOAc/MeOH; 97: 3)gave 4 (160 mg, 53%) as ˜1:1 mixture of diastereoisomers: ¹H NMR δ 0.04(s, 6,2×CH₃), 0.88 (s, 9, t-Bu), 1.18-1.34 (complex m, 6, 2×CH₃),2.05-2.21 (m, 2, H2′,2″), 3.50-3.63 (complex m, 4, H4′,4″, CH₂),3.70-3.82 (m, 1, H3′), 4.31-4.40 (m, 2, H1′,1″), 4.72-4.78 & 4.88-4.93(2×m, 1, CH), 5.99 & 6.06 (2×br s, 2, NH₂), 7.86 & 8.04 (2×s, 1, H8),8.37 (s, 1, H2); ¹³C NMR δ −5.1(2×CH₃), 15.7 & 15.9 (CH₃), 18.6 (t-Bu),20.7 & 21.0 (CH₃), 26.2 (t-Bu), 32.5 & 32.7 (C2′), 41.0 & 41.1 (C1′),61.0 & 62.1 (CH₂), 65.5 & 66.1 (C4′), 73.6 & 74.8 (C3′), 100.2 & 100.5(CH), 119.9 (C5), 141.1 & 142.2 (C8), 150.5 (C4), 152.1 & 152.5 (C2),155.3 (C6); MS (APCI) m/z 410 (100, MH⁺). Anal. Calcd for C₁₉H₃₅N₅O₃Si(409.60): C, 55.71; H, 8.61; N, 17.10.

9-[-3-O-(1-Ethoxyethyl)-3,4-dihydroxybutyl] adenine (5)—Procedure A

TBAF/THF (0.88 mL, 1M) was added to a solution of 4 (180 mg, 0.44 mmol)in dry THF (6 mL) and the mixture was stirred at ambient temperature for20 min. Volatiles were evaporated and the residue was columnchromatographed (EtOAc/MeOH; 78:12) to give 5 (120 mg, 92%) as ˜1:1mixture of diastereoisomers: ¹H NMR δ 1.21-1.25 (complex m, 3, CH₃),1.35 & 1.39 (d, J=5.3 Hz, 3, CH₃), 2.05-2.25 (m, 2, H2′,2″), 3.53-3.82(complex m, 5, H3′,4′,4″, CH₂), 4.32-4.39 (m, 2, H1′,1″), 4.69 &4.88-4.87 (q, J=5.3 Hz, 1, CH), 5.88 & 5.92 (2×br s, 2, NH₂), 7.82 &7.94 (2×s, 1, H8), 8.36 (s, 1, H2); ¹³C NMR δ 15.5 & 15.7 (CH₃), 20.4 &20.6 (CH₃), 32.5 & 32.7 (C2′), 40.0 & 41.1 (C1′), 60.9 & 62.4 (CH₂),64.9 & 65.9 (C4′), 73.2 & 78.9 (C3′), 99.6 & 101.5 (CH), 119.7 (C5),140.8 & 141.5 (C8), 150.5 (C4), 152.8 & 153.1 (C2), 155.6 & 155.7 (C6);MS (APCI) m/z 296 (100, MH⁺). Anal. Calcd for C₁₃H₂₁N₅O₃ (295.34): C,52.87; H, 7.17; N, 23.71.

Methyl 4-(Adenin-9-yl)-2-hydroxybutanoate (6)—Procedure B

To a suspension of 5 (90 mg, 0.31 mmol) in CH₃CN/CCl₄/H₂O (1:1:1.5; 1.5mL), NaHCO₃ (161 mg, 0.88 mmol), NaIO₄ (353 mg, 1.65 mmol) and RuCl₃(trace) were added. The mixture was stirred at ambient temperature for48 h until no starting material was detected on TLC. Then water (5 mL)and CHCl₃ (4 mL) were added, the two layers were separated and waterphase was washed with CHCl₃ (3 mL). The aqueous layer was acidified withHCl to pH 4 and applied on a column of Dowex 50 W ×2 (H⁺). Column waswashed with 200 mL of water then product was eluted with 2.5% NH₄OH/H₂O.The combined UV-absorbing ammonia eluate was evaporated and coevaporatedwith MeOH (2×). The residue was dissolved in MeOH (5 mL) and a solutionof CH₂N₂ in diethyl ether was added until yellow color of diazomethanewas maintained during several minutes. The solution was concentrated andcolumn chromatographed (CHCl₃/MeOH; 95:5) to give 6 (31 mg, 41%) as awhite solid with data identical as reported. To avoid formation ofby-product 6′ it is imported to keep desired amount of NaHCO₃ inreaction mixture.

4-(Adenin-9-yl)-2-hydroxybutanoic acid (7)—Procedure C

NaOH/H₂O (1 mL. 0.1 M) was added to a solution of 6 (10 mg, 0.04 mmol)in MeOH/H₂O (2.0 mL). The mixture was stirred at ambient temperature for6 h until no starting material was detected on TLC. Then reactionmixture was acidified with HCl to pH ˜4 and applied on a column of Dowex50 W×2 (H⁺). Column was washed with water (100 mL) and then product waseluted with 2.5% NH₄OH. The combined UV-absorbing ammonia eluate wasevaporated to give 7 as a ammonium salt (7.6 mg, 75%) with data asreported.

9-(3,4-O-Di-t-Butyldimethylsilyl-3,4-dihydroxybutyl)adenine (8)

TBDMS-C1 (593 mg, 3.92 mmol) and imidazole (534 mg, 7.85 mmol) wereadded to a stirred solution of 2 (350 mg, 1.57 mmol) in dry DMF (8 mL),and the mixture was stirred at ambient temperature overnight. Thenreaction mixture was partitioned between EtOAc/NH₄Cl/H₂O. The waterlayer was extracted with EtOAc. The combined organic phase was washed(brine), dried (Na₂SO₄), and evaporated. Column chromatography (CHCl₃→3%MeOH/CHCl₃) gave 8 (610 mg, 86%): ¹H NMR δ 0.04 (s, 6, 2×CH₃), 0.09 (s,6, 2×CH₃), 0.88 (s, 9, t-Bu), 0.92 (s, 9, t-Bu), 2.02-2.07 (m, 1, H2′),2.21-2.26 (m, 1, H2″), 3.46 (dd, J=6.8, 10.0 Hz, 1, H4′), 3.59 (dd,J=5.2, 10.0 Hz, 1, H4″), 3.78-3.81 (m, 1, H3′), 4.32-4.45 (m, 2,H1′,1″), 5.96 (br s, 2, NH₂), 7.84 (s 1, H8), 8.42 (s, 1, H2); MS (APCI)m/z 452 (100, MH⁺). Anal. Calcd for C₂₁, H₄₁N₅O₂Si₂₁ (451.76): C, 55.83;H, 9.15; N, 15.50.

9-(3-O-t-Butyldimethylsilyl-3,4-dihydroxybutyl)adenine (9)

Compound 8 (400 mg, 0.887 mmol) was added to a solution ofCH₃CO₂H/H₂O/THF (13:7:3; 8 mL) and the mixture was stirred at ambienttemperature until more polar spot of compound 2 starting to appear onTLC. Then reaction mixture was partitioned (EtOAc//NaHCO₃/H₂O) and theaqueous layer was extracted with next portion of EtOAc. The combinedorganic phase was washed (NaHCO₃, brine), dried (Na₂SO₄), evaporated andcolumn chromatographed (CHCl₃→4% MeOH/CHCl₃) to give recovered 8 (140mg, 35%) and 9 (155 mg, 52%): ¹H NMR δ 0.04 (s, 6, 2×CH₃), 0.88 (s, 9,t-Bu), 2.16-2.19 (m, 2, H2′,2″), 3.61 (dd, J=4.5, 11.4 Hz, 1, H4″), 3.65(dd, J=5.2, 11.4 Hz, 1, H4″), 3.88 (q, J=5.2 Hz, 1, H3′), 4.26-4.36 (m,2, H1′,1″), 6.17 (br s, 2, NH₂), 7.80 (s, 1, H8), 8.29 (s, 1, H2); ¹³CNMR δ −4.4 & 4.1 (CH₃), 18.5 (t-Bu), 26.2 (1-Bu), 34.5 (C2′), 40.8(C1′), 65.5 (C4′), 70.6 (C3′), 119.8 (C5), 140.7 (C8), 150.4 (C4), 153.0(C2), 155.5 (C6); MS (APCI) m/z 338 (100, MH⁺). Anal. Calcd forC₁₅H₂₇N₅O₂Si (337.50): C, 53.38; H, 8.06; N, 20.75.

Methyl 3-(Adenin-9-yl)propionate (6′)

Treatment of 9 (50 mg, 0.148 mmol) by procedure B (columnchromatography: CHCl₃/MeOH 97:3) gave 6′ (9 mg, 27%) with data asreported.

3-(Adenin-9-yl)propionic acid (7′)

Treatment of 6′ (10 mg, 0.045 mmol) by procedure C (columnchromatography: CHCl₃/MeOH 97:3) gave 6′ (7.3 mg, 78%) with data asreported.

Example 2 DZ2002 Mediates Immunosuppressive Effects

Materials and Methods

Reagents

AdoHcy hydrolase inhibitors DZ2002 was synthesized at DiazymeLaboratories. Con A (Concanavalin A), LPS (Escherichia coli 055:B5) andSac (Staphylococcus aureus Cowan strain1) were obtained from Pansorbin®cells, Biosciences, inc.(La Jolla., CA 92039, USA). RPMI 1640 and fetalbovine serum (FBS) were obtained from GIBCO. Purified rat anti-mouseIL-10, IL-12p70, IL-12p40, IFN-γ and biotinylated anti-mouse IL-10,IL-12p70, IL-12p40, IFN-γ, FITC-anti-mouse-CD 11 b (Mac-1), Phycorythrin(PE)-anti-mouse I-Ad, PE-anti-human-CD14, PE-anti-human-ABC,PE-anti-human-DR, PE-anti-human-CD80 and PE-anti-human-CD86 werePharmingen products. Thioglycollate (TG) is available fromSigma-Aldrich.

Animal

Inbred BALB/C mice, 6˜8 weeks of age, were provided by ShanghaiExperimental Animal Center of Chinese Academy of Sciences withCertificate No. 99-003. The mice were housed in specific pathogen-free(SPF) conditions with room temperature of 24±2° C., 12 hr light/darkcycle, and provided with sterile food and water ad libitum.

Cells

Spleens from Balb/c mice were aseptically removed, pooled, and singlecell suspensions prepared in PBS. Erythrocytes were lysed by treatmentwith Tris-buffered ammonium chloride (0.155 M NH₄CL, 0.0165 M Tris, PH7.2). Mononuclear cells were washed with PBS and resuspended inRPMI-1640 media supplemented with benzylpenicillin 100000 U·L⁻¹, andstreptomycin 100 mg·L⁻¹. The cell viability and concentration weredetermined by trypan blue exclusion.

Peritoneal exudate cells were induced in BALB/C mice by anintraperitoneal injection of 0.5 ml of 3% TG. After 4 days, theperitoneal exudates cells were harvested by sterile lavage.

THP-1 (American Type Culture Collection, Manassas, Va.) is a humanmonocytic leukemia. THP-1 cells were maintained in suspension culture inRPMI1640 medium supplemented with 10% FBS. Cultures were maintained at37° C. in a humidified atmosphere of 5% CO₂ in air and were subcultureat 1/10 dilution every 5-6 days.

[³H]-thymidine Incorporation to the Splenic Lymphocytes

Mouse splenic lymphocytes were cultured in vitro in RPMI1640supplemented with 10% FBS. Cells were incubated in a 96-well plate at1×10⁵ cells/200 μl/well in a humidified CO₂ incubator at 37° C. for 48hours with 5 μg/ml of Con A or 10 μg/ml LPS in the presence or absenceof various concentrations of DZ2002. After 40 hour incubation, cellswere pulsed with 0.5 μCi/well of [³H]-thymidine and cultured for another8 hours. The cells were then harvested onto glass fiber filters and theincorporated radioactivity was counted using a Beta Scintillator(MicroBeta Trilux, PerkinElmer Life Sciences).

MTT Assay of the Splenic Lymphocytes

Cytotoxicity was assessed with MTT assay. Mouse splenic lymphocytes wereincubated in a 96-well plate at 9×10⁴cells/180 μL/well in a humidifiedCO₂ incubator at 37° C. for 48 hours in the presence or absence ofvarious concentrations of DZ2002. Fifteen (15) μl of 5 mg/ml of MTT waspulsed 4 h prior to end of the culture (total 190 μl), and then 80 μlsolvent (10% SDS, 50% N,N-dimethy formamide, PH7.2) was added. Incubatefor 7 h and read OD₅₉₀ at a microplate reader (Bio-rad Model 550 Japan).

Cytokine production

Murine splenic mononuclear cells (5×10⁶) were cultured in 24-well platesin a volume of 2 ml/well in the presence of Sac (1:10000), ConA (5ug/ml) or LPS (10 ug/ml) in the presence or absence of variousconcentrations of DZ2002. After 24 h, cell-free supernatant wascollected and frozen at −20° C. The concentrations of IL-12p40,IL-12p70, IL-10 and TNF-α were determined in an ELISA specific formurine cytokines.

Murine peritoneal exudate cells (6.25×10⁵) were cultured in 24-wellplates in a volume of 1 ml/well for 2 hours. In adherent cells werewashed by ice cold RPMI 1640 and adherent cells were culture in a volumeof 2 ml/well in the presence of IFN-γ (2.5 ng/ml) and LPS (1 μg/ml) inthe presence or absence of various concentrations of DZ2002. After 24 h,cell-free supernatant was collected and frozen at −20° C. Theconcentrations of IL-12p40, IL-12p70, IL-10 and TNF-α: were determinedby ELISA.

THP-1 cells (6×10⁵) were cultured in 24-well plates in a volume of 2ml/well in the presence of 1.2% and in the presence or absence ofvarious concentrations of DZ2002. After 24 h, IFN-γ (500 U/ml) was addedand another 16 h later, LPS (1 μg/ml) was added. Cell-free supernatantwas collected after 24 h and frozen at −20° C. The concentrations ofIL-12p40, IL-12p70, IL-10 and TNF-α were determined for ELISA.

Quantitative Hemolysis of Sheep Red Blood Cells (QHS) Assay

Female Babl/c mice were immunized by intraperitoneal injection with 0.2ml of 16.7% of SRBC on day 4. Vehicle, Dexamethasone and DZ2002 wereadministrated on each group (n=6) by intraperitoneal injection on 7consecutive days of 1-7. On day 8, mice were sacrificed and made a mixedsuspension of spleen cells of 2×10⁶ cells/ml. 1 ml of cell suspensionwas incubated with 1 ml of 0.5% SRBC and 1 ml of 1:10 dilution of guineapig complement for 1 h at 37° C., then centrifuged (3 min, 3000 g) anddetermined the supernatant hemolysis at 413 nm, according to Simpson etal, J. Immunol. Methods., 21(1-2):159-65.(1978) with some modifications.Each group was triplicated.

Mixed Lymphocyte Reaction (MLR) Proliferation Assay.

Balb/c mouse spleen cells were prepared in 10⁷ cells/ml suspension,cultured 2 h with 50 μg/ml of mitomycin. Then cells were washed andcultured together with fresh C57/B6 mice splenocytes equally in a finalconcentration of 1.0×10⁶ cells/ml in the presence or absence of variousconcentrations of DZ2002. After 48 hour incubation, cells were pulsedwith 0.5 μCi/well of [³H]-thymidine and cultured for another 24 hours.The cells were then harvested onto glass fiber filters and theincorporated radioactivity was counted using a Beta Scintillator(MicroBeta Trilux, PerkinElmer Life Sciences).

DNFB-Induced Delayed Type Hypersensitivity (DTH) Response

Female Balb/c mice were sensitized with 20 μl of 0.6% DNFB dissolved inacetone-olive oil (4:1) on each hind foot on day 0 and 1. On day 7 micewere challenged with 10 μl of 0.5% DNFB on both sides of left ear,methods according to Phanuphak (1974) with some modifications. Vehicle,CsA, and DZ2002 (1, 3, 10 mg/kg) were administrated on each group (n=10)by intraperitoneal injection on 1 hour before and 12 hours, 24 hoursafter the challenge. Ear swelling was expressed as difference betweenthe weight of the left and right ear patches made by a specific 8-mmpunch 30 h after the challenge.

Flow Cytometry

Murine peritoneal exudate cells or THP-1 cells were washed in cold PBS(staining buffer, containing 0.1% NaN₃, 1% FBS, PH 7.2). Cells wereresuspended at 2.0×10⁷/ml in cold staining buffer. Optimalconcentrations of each fluorochrome-labeled antibody were added to 50 μLcells. Fc receptors were blocked using 10 μL normal mouse serum. Cellswere incubated in the dark at 4° C. for 30 min, washed twice with 2.0 mLstaining buffer and resupended in 0.5 mL of PBS, PH 7.2. Cells werestored in the dark at 4° C. and analyzed on a FACScan flow cytometer(Becton Dickinson, San Jose, Calif.). Data were analyzed by means ofCellQuest™ Software (Becton Dickinson, San Jose, Calif.).

Statistical Analysis

Results were expressed as x±s, independent two-tailed t-test wasperformed and P values less than 0.05 were considered to be significant.Each experiment was repeated at least three times.

RESULTS

Inhibition of [³H]-Thymidine Incorporation to the Splenic Lymphocytes byAdoHcy Hydrolase Inhibitors

After 48 h of culture, DZ2002 (0.1-10 μmol·L⁻¹) have no effects on thelymphocytes proliferation induced by ConA. DZ2002 (10 μmol·L⁻¹)inhibited lymphocytes proliferation induced by LPS.

Effect of DZ2002 on IL-10, IL-12P40 and IL-12P70 Production from SacStimulated Murine Splenocytes

Sac stimulation induced marked increasing of IL-10, IL-12P40 and IFN-γproduction from murine splenocytes compared with resting splenocytes.DZ2002 (μmol·L⁻¹) dose dependently inhibited IL-12P40, IL-12P70 andTNF-α release, but have no effect on IL-10 production from Sacstimulated splenocytes. (data not shown).

Effects of DZ2002 on Quantitative Hemolysis of Sheep Red Blood Cells(QHS) Assay

Quantitative hemolysis of SRBC is a model of primary antibody productionin response to antigenic stimulation. As FIG. 3 shows, consecutively7-day intraperitoneal injection of DZ2002 inhibited 24.5 and 18.4% ofQHS at doses of 0.08 and 2 mg/kg respectively, compared with 38.1% ofthat of 5 mg/kg Dethamethasone (p<0.05 for All experiment groupscompared with Vehicle control group) (FIG. 1).

DZ2002 Suppress T Cell Proliferation in Mixed Lymphocyte Reaction

Mitomycin-treated Balb/c (H-2^(d)) spleen cell were applied asallogeneic stimulator to C57BL/6(H1-2b) spleen cells proliferation.DZ2002 had a strong suppression to MLR with 40.2, 36.9 and 42.3% atdoses of 0.1, 1 and 10 μmol/L respectively for 3-day culture. (FIG. 2).

DZ2002 have no Cytotoxicity in Spleen Cell

In two days of culture, 0.1-10 mmol/L DZ2002 showed no cytotoxicity tospleen cells. The OD values of cells incubated with DZ2002 have nodifference with that of the control. (FIG. 3).

DZ2002 Reversed the Suppression of Mouse DTH Response Induced by EthanolConsumption

Nine mice were prepared for each group. Mice were sensitized with 0.5%DNFB solution (20 ul) in absolute acetone/olive oil (4:1) on each hindfoot on day 0 and 1. Five days after initial sensitization, mice werechallenged with 0.2% DNFB (10 ul) on both sides of left ear under lightMetofane anesthesia. The right ear was treated with vehicle alone.DZ2002 were orally administered to the mice 1 h before DNFB challenge.The degree of ear swelling was measured 24 h after challenge using a earpuncher and an analytic balance to measure the weight (mg). Results wereexpressed as the difference between the weight of the left and the rightear. Spleens were taken from four mice in each group after themeasurement of the ear swelling and frozen until analysis.

Effects of DZ2002 on the Expression of MHC-II on Resident and TG InducedPeritoneal Cells

MHC-II expression by peritoneal macrophages was assessed following a48-h incubation with media alone or with IFN-γ at 100 U/ml. Cellsincubated with IFN-γ in the present of 0.1 and 1 μmol/L DZ2002 enhancethe levels of MHC-IL expression of resident peritoneal cells and 10μmol/L DZ2002 reduce the levels of MHC-II expression.(data not shown) Asreflected in the TG induced peritoneal cells, 1 and 10 μmol/L DZ2002decrease the Mac-1⁺ percentage when incubate with media alone. And thelevel of MHC-II expression of cells incubated with IFN-γ wasdose-dependently decreased in the presence of 0.1-10 μmol/L DZ2002.(data not shown).

Effect of DZ2002 on IL-10, IL-12P40 and TNF-α Production from TG InducedPeritoneal Cells

Cytokines produced by peritoneal macrophages were assessed following a24-h incubation with IFN-γ at 25 U/ml and LPS at 1 μg/ml. Residentperitoneal cells produce low levers of cytokines except for some IL-10with incubated with IFN-γ and LPS (data not shown). As for TG inducedperitoneal macrophages, DZ2002 inhibited IL-12P40 and TNF-α release, buthave no effect on IL-10 production in the dose of 0.1-10 μmol/L (FIG.5).

DZ2002 Inhibits Expression of MHC-II, CD80 and CD86 on THP-1 Cells

MHC-II, CD80 and CD86 expression by THP-1 cells was assessed following a48-h incubation with media alone or with IFN-γ at 100 U/ml. Cellsincubated with IFN-γ in the present of 10 μmol/L DZ2002 modestly reducethe levels of MHC-II expression of THP-1 cells and 0.1-10 μmol/L DZ2002reduce the levels of CD80 and CD86 expression by an dose-dependentlyway. (FIG. 6A-C).

Effect of DZ2002 on IL-10, IL-12P40 and TNF-α Production from THP-1Cells

Cytokines produced by THP-1 cells were assessed following a 24-hincubation with IFN-γ at 500 U/ml and LPS at 1 μg/ml. As FIG. 7 shows,DZ2002 inhibited IL-12P40 and TNF-α release, in the dose of 0.1-10mmol/L.

The above examples are included for illustrative purposes only and arenot intended to limit the scope of the invention. Many variations tothose described above are possible. Since modifications and variationsto the examples described above will be apparent to those of skill inthis art, it is intended that this invention be limited only by thescope of the appended claims.

1-50. (canceled)
 51. A method for treating multiple sclerosis in amammal, which method comprises administering to a mammal having multiplesclerosis, an effective amount of a compound or a pharmaceuticallyacceptable salt thereof, having the formula (I):

wherein Z is selected from the group consisting of carbon and nitrogen,R1 and R2 are the same or different, and are selected from the groupconsisting of hydrogen, hydroxy, unsubstituted alkyl, substituted alkyl,cycloalkyl, alkenyl, alkoxy, amino, aryl, heteroaryl, and halogen; R3and R4 are the same or different and are selected from the groupconsisting of hydrogen, unsubstituted alkyl, substituted alkyl, acetyl,alkenyl, aryl, and heteroaryl; X is selected from the group consistingof oxygen, —NH, and sulfur; and Y is selected from the group consistingof hydrogen, a C₁₋₁₀ unsubstituted alkyl group, a C₁₋₁₀ substitutedalkyl group, alkenyl, vinyl, aryl, and heteroaryl, thereby treating saidmultiple sclerosis in said mammal.
 52. The method of claim 51, whereinsaid mammal is human.
 53. The method of claim 51, wherein said compoundis not (4-adenine-9-yl)-2-hydroxybutanoic acid.
 54. The method of claim51, wherein said compound is Methyl 4-(Adenin-9-yl)-2-hydroxybutanoateor a pharmaceutically acceptable salt thereof.
 55. The method of claim51, wherein the β carbon relative to the COXY group of said compound hasa R configuration.
 56. The method of claim 51, wherein R1, R2, R3, andR4 are hydrogen, X is oxygen, and Y is a C₁₋₁₀ unsubstituted alkyl or aC₁₋₁₀ substituted alkyl.
 57. A method for inhibiting lymphocyteproliferation, inhibiting production and/or release of IL-12P40,IL-12P70 and TNF-α or inhibiting primary antibody production in amammal, which method comprises administering to a mammal an effectiveamount of a compound or a pharmaceutically acceptable salt thereof,having the formula (I):

wherein Z is selected from the group consisting of carbon and nitrogen,R1 and R2 are the same or different, and are selected from the groupconsisting of hydrogen, hydroxy, unsubstituted alkyl, substituted alkyl,cycloalkyl, alkenyl, alkoxy, amino, aryl, heteroaryl, and halogen; R3and R4 are the same or different and are selected from the groupconsisting of hydrogen, unsubstituted alkyl, substituted alkyl, acetyl,alkenyl, aryl, and heteroaryl; X is selected from the group consistingof oxygen, —NH, and sulfur; and Y is selected from the group consistingof hydrogen, a C₁₋₁₀ unsubstituted alkyl group, a C₁₋₁₀ substitutedalkyl group, alkenyl, vinyl, aryl, and heteroaryl, thereby inhibitinglymphocyte proliferation, inhibiting production and/or release ofIL-12P40, IL-12P70 and TNF-α or inhibiting primary antibody productionin said mammal.
 58. The method of claim 57, wherein said mammal ishuman.
 59. The method of claim 57, wherein said compound is not(4-adenine-9-yl)-2-hydroxybutanoic acid.
 60. The method of claim 57,wherein said compound is Methyl 4-(Adenin-9-yl)-2-hydroxybutanoate or apharmaceutically acceptable salt thereof.
 61. The method of claim 57,wherein the β carbon relative to the COXY group of said compound has a Rconfiguration.
 62. The method of claim 57, wherein lymphocyteproliferation is inhibited in said mammal.
 63. The method of claim 57,wherein production and/or release of IL-12P40 or IL-12P70 is inhibitedin said mammal.
 64. The method of claim 57, wherein primary antibodyproduction is inhibited in said mammal.
 65. The method of claim 57,wherein R1, R2, R3, and R4 are hydrogen, X is oxygen, and Y is a C₁₋₁₀unsubstituted alkyl or a C₁₋₁₀ substituted alkyl.